Liposomes are closed vesicles composed of lipid bilayers and can carry various substances in the inside aqueous phase or between the lipid membranes. Therefore, investigations have been undertaken to utilize as drug delivery systems liposomes with fat-soluble or amphipathic substances such as physiologically active substances (drugs etc.) entrapped therein. For example, missile therapy has been developed wherein cancer cells are selectively attacked by an anticancer agent-containing liposomes combined with a cancer surface-specific antibody.
Liposomes with a physiologically active substance entrapped therein are usually prepared by dissolving lipids which have ability to form liposome membrane, for example phospholipids, cholesterols, acidic phospholipids, etc., in an organic solvent, distilling off the solvent to some extent from this lipid solution to obtain a lipid mixture, adding an aqueous solution of the physiologically active substance to the lipid mixture and stirring the mixture vigorously. The lipid mixture according to the above method is a dense solid and can not be fully hydrated when mixed with the aqueous solution of physiologically active substance with stirring. The hydration becomes more difficult with increasing lipid concentration of the lipid mixture. Therefore, such a conventional method has various problems. More specifically, the spacing between the lipid membranes, which is very small, results in a reduced efficiency in encapsulating the physiologically active substances or reduces the encapsulating volume of the liposomes. The loss of lipids increases, i.e., the ratio of the lipids consumed for forming liposomes to the total amount of lipids used reduces. Further, defects in the lipid membrane, which frequently occur, result in reduced stability and leakage of the physiologically active substance entrapped.
In the specification, the encapsulating efficiency means the ratio of the amount of physiologically active substance entrapped to the total amount of physiologically active substance used (V/V). The encapsulating volume means the total volume of the inside aqueous phase of liposomes formed by one mole of lipids.
Furthermore, the above conventional method, when practiced industrially for mass production of liposomes aggravates the above problems, encounters difficulty in selecting the conditions for the distillation of solvent and requires a specific device for vigorous stirring to entail an increased cost. Therefore, the above conventional method has great difficulties in placing liposome compositions into practical use.
In order to overcome the above problems, various methods have been proposed.
For example, Unexamined Japanese Patent Publication No. 7933/1985 discloses a method for preferring liposomes at a higher temperature than in conventional method. By this method, however, thermolabile drugs can not be entrapped in liposomes.
Unexamined Japanese Patent Publication No. 7932/1985 discloses a method for preparing liposomes using lipids having liposome membrane forming ability in combination with polyols. However, the use of polyols leads to difficulty in homogeneously dispersing cholesterols, which are important components of the lipid membrane of liposomes, therefore gives unstable liposome membranes and permits leakage of physiologically active substances entrapped.
Unexamined Japanese Patent Publication No. 152531/1987 discloses a method for preparing liposomes comprising spray-drying into a powder a solution of lipids having liposome membrane forming ability with a spray drier and mixing the powder with an aqueous solution of physiologically active substances. However, since this powder is likely to agglutinate, it has the drawback of being difficult to hydrate. Therefore, the liposomes prepared by dissolving this powder in water is low in ability to encapsulate physiologically active substances. This method further permits an increased loss of lipids.
Furthermore, Examined Japanese Patent Publication No. 52724/1987 discloses a method for preparing liposomes carrying a pysiologically active substance, which comprises freeze-drying a solution of liposomes having liposome membrane forming ability and physioligically active substance into a powder and dissolving the powder in water. However, this method requires expensive equipment for freeze-drying, and is time-consuming to distill the solvent off, hence a greatly increased production cost.
Besides the above methods, a reverse-phase evaporation method is known, which comprises adding an aqueous solution of physiologically active substance to a solution of lipids having liposome membrane forming ability, preparing liposomes by ultrasonic emulsification and distilling the solvent. However, this method has the problem that it is difficult to distill the solvent off thoroughly and that the remaining solvent tends to give toxicity to a pharmaceutical preparation of liposomes or to inhibit the formation of liposome membrane.